F9 6 1000 lex rotor

Htt^ex1Q46 was purified as described previously (8 (link)). Htt^ex1Q46 was grown in BL21 cells to an OD₆₀₀ of 0.6 and induced with 1mM IPTG (GoldBio) overnight at 16°C. Cells were spun down at 7,000rpm (Thermo Scientific F9–6×1000 LEX rotor) and resuspended in 15mM Tris-HCl buffer, pH 8.0. Lysozyme was added for lysis, and cells were tumbled at 4°C for 45min. Lysates were spun down at 12,000rpm (Thermo Scientific F20–12×50 LEX rotor) for 10min and the supernatant added to 3mL of nickel beads (GoldBio) per 100mL of lysate and tumbled for 4 h at 4°C. Beads were washed 3 times with 15mM Tris-HCl, pH 8.0 and 3 more times with wash buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 1mM PMSF, 1mM EDTA). Protein was eluted off beads by tumbling overnight at 4°C in 25mL wash buffer with 250mM imidazole. Protein was dialyzed into ThT assay buffer (20mM Tris-HCl pH 8.0, 50mM NaCl, 2mM CaCl₂).

Ataxin-3^Q55 was purified as described previously (63 (link)). GST-ATXN3^Q55 was grown in BL21 cells to an OD₆₀₀ of 0.6 and induced with 1mM IPTG (GoldBio) overnight at 16°C. Cells were spun down at 7,000rpm (Thermo Scientific F9–6×1000 LEX rotor) and resuspended in NETN buffer (50mM Tris, pH 7.5, 150mM NaCl, 0.5% IGEPAL CA-630) containing aprotinin (GoldBio), leupeptin (Alfa Aesar), PMSF (Grainger), and DTT (Sigma). For lysis, cells were tumbled at 4°C for 30min and sonicated 3 times for 30s. Lysates were spun down at 12,000 rpm (Thermo Scientific F20–12×50 LEX rotor) for 10min and the supernatant was added to glutathione sepharose beads (GoldBio) and tumbled at 4°C for over 1h. Beads were then washed 3 times with NETN and washed 3 more times with PBS. Protein was eluted off the beads by incubating with HRV 3c protease overnight at 4°C.

Thio-polyQ45 was grown in BL21 cells to an OD₆₀₀ of 0.6 and induced with 1mM IPTG (GoldBio) overnight at 16°C. Cells were spun down at 7,000rpm (Thermo Scientific F9–6×1000 LEX rotor) and resuspended in Buffer A (50mM Tris, pH 7.5, 1M NaCl, 0.5% Triton X-100). Lysozyme was added for lysis, and cells were tumbled at 4°C for 30min. Lysates were spun down at 12,000rpm (Thermo Scientific F20–12×50 LEX rotor) for 10min and the supernatant added to 500μL of nickel beads (GoldBio) per 1L of cells and tumbled for 3 h at 4°C. Beads were washed 3 times with Buffer A, 3 more times with Buffer B (50mM Tris, pH 7.5, 1M NaCl, 20mM imidazole), and 3 more times with Buffer C (50mM Tris, pH 7.5, 150mM NaCl, 20mM imidazole). Protein was eluted off beads by tumbling overnight at 4°C in 5 mL Buffer C with 300mM imidazole. Protein was dialyzed into ThT assay buffer (20mM Tris-HCl pH 8.0, 50mM NaCl, 2mM CaCl₂).